Ningning Zhuang

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

Crystallization and preliminary X-ray data analysis of a DJ-1 homologue from Arabidopsis thaliana (AtDJ-1D).

A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, β = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

dicPAH and dicPAH bind by molecular sieving (View Interaction: 1, 2, 3, 4)

Unusual NADPH conformation in the crystal structure of a cinnamyl alcohol dehydrogenase from Helicobacter pylori in complex with NADP(H) and substrate docking analysis

HpCAD and HpCAD bind by x‐ray crystallography (View interaction)

Structural basis of a novel activity of bacterial 6‐pyruvoyltetrahydropterin synthase homologues distinct from mammalian 6‐pyruvoyltetrahydropterin synthase activity

Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn(2+) binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.

Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei

Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32 M MgCl2, 0.08 M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a=97.11, b=135.92, c=46.03 Å. Diffraction data were collected to 2.08 Å resolution.

Purification, crystallization and crystallographic analysis of Dictyostelium discoideum phenylalanine hydroxylase in complex with dihydrobiopterin and FeIII

Dictyostelium discoideum phenylalanine hydroxylase (DicPAH; residues 1-415) was expressed in Escherichia coli and purified for structural analysis. Apo DicPAH and DicPAH complexed with dihydrobiopterin (BH(2)) and Fe(III) were crystallized using 0.06 M PIPES pH 7.0, 26%(w/v) PEG 2000 by the hanging-drop vapour-diffusion method. Crystals of apo DicPAH and the DicPAH-BH(2)-Fe(III) complex diffracted to 2.6 and 2.07 A resolution, respectively, and belonged to space group P2(1), with unit-cell parameters a = 70.02, b = 85.43, c = 74.86 A, beta = 110.12 degrees and a = 70.97, b = 85.33, c = 74.89 A, beta = 110.23 degrees , respectively. There were two molecules in the asymmetric unit. The structure of DicPAH has been solved by molecular replacement.

Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) from Synechococcus sp. PCC 7942.

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacterium Synechococcus sp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 M bis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 171.35, b = 77.99, c = 53.77 Å, β = 90.27°.

Structural analysis of E. coli 6-carboxytetrahydropterin synthase

1 Gyeongsang National University, Department of Microbiology, School of Medicine, Jinju, Republic of Korea, 2 Gyeongsang National University, Department of Biomedical Sciences, Graduate School, Jinju, Republic of Korea, 3 Gyeongsang National University, Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Jinju, Republic of Korea, 4 School of Biological Sciences, Inje University, Kimhae, Republic of Korea

Structural insights into the dual substrate specificities of mammalian and Dictyostelium dihydropteridine reductases toward two stereoisomers of quinonoid dihydrobiopterin

DHPR binds to DHPR by X‐ray crystallography (View interaction)